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Summary The earliest stages of Alzheimer's disease (AD) are characterized by the formation of mature tangles in the entorhinal cortex and disorientation and confusion navigating familiar places. The medial entorhinal cortex (MEC) contains specialized neurons called grid cells that form part of the spatial navigation system. Here we show in a transgenic mouse model expressing mutant human tau predominantly in the EC that the formation of mature tangles in old mice was associated with excitatory cell loss and deficits in grid cell function, including destabilized grid fields and reduced firing rates, as well as altered network activity. Overt tau pathology in the aged mice was accompanied by spatial memory deficits. Therefore, tau pathology initiated in the entorhinal cortex could lead to deficits in grid cell firing and underlie the deterioration of spatial cognition seen in human AD.

BackgroundHaving the apolipoprotein E4 (APOE-ϵ4) allele is the strongest genetic risk factor for the development of Alzheimer’s disease (AD). Accumulation of amyloid beta (Aβ) in the brain is influenced by APOE genotype. Transgenic mice co-expressing five familial AD mutations (5xFAD) in the presence of human APOE alleles (ϵ2, ϵ3 or ϵ4) exhibit APOE genotype-specific differences in early Aβ accumulation, suggesting an interaction between APOE and AD pathology. Whether APOE genotype affects Aβ-plaque-associated neuroinflammation remains unclear. In the current study, we address the role of APOE genotype on Aβ-associated microglial reactivity in the EFAD transgenic mouse model.MethodsWe analyzed Aβ-induced glial activation in the brains of 6-month-old EFAD transgenic mice (E2FAD, E3FAD and E4FAD). Region-specific morphological profiles of Aβ plaques in EFAD brain sections were compared using immunofluorescence staining. We then determined the degree of glial activation in sites of Aβ deposition while comparing levels of the inflammatory cytokine Interleukin-1β (IL-1β) by ELISA. Finally, we quantified parameters of Aβ-associated microglial reactivity using double-stained EFAD brain sections.ResultsCharacterization of Aβ plaques revealed there were larger and more intensely stained plaques in E4FAD mice relative to E2FAD and E3FAD mice. E4FAD mice also had a greater percentage of compact plaques in the subiculum than E3FAD mice. Reactive microglia and dystrophic astrocytes were prominent in EFAD brains, and primarily localized to two sites of significant Aβ deposition: the subiculum and deep layers of the cortex. Cortical levels of IL-1β were nearly twofold greater in E4FAD mice relative to E3FAD mice. To control for differences in levels of Aβ in the different EFAD mice, we analyzed the microglia within domains of specific Aβ deposits. Morphometric analyses revealed increased measures of microglial reactivity in E4FAD mice, including greater dystrophy, increased fluorescence intensity and a higher density of reactive cells surrounding cortical plaques, than in E3FAD mice.ConclusionsIn addition to altering morphological profiles of Aβ deposition, APOE genotype influences Aβ-induced glial activation in the adult EFAD cortex. These data support a role for APOE in modulating Aβ-induced neuroinflammatory responses in AD progression, and support the use of EFAD mice as a suitable model for mechanistic studies of Aβ-associated neuroinflammation.

The apolipoprotein E4 allele is the strongest genetic risk factor for developing late-onset Alzheimer's disease, and may predispose individuals to Alzheimer's-related cognitive decline by affecting normal brain function early in life. To investigate the impact of human APOE alleles on cognitive performance in mice, we trained 3-mo-old APOE targeted replacement mice (E2, E3, and E4) in the Barnes maze to locate and enter a target hole along the perimeter of the maze. Long-term spatial memory was probed 24 h and 72 h after training. We found that young E4 mice exhibited significantly impaired spatial learning and memory in the Barnes maze compared to E3 mice. Deficits in spatial cognition were also present in a second independent cohort of E4 mice tested at 18 mo of age. In contrast, cognitive performance in the hidden platform water maze was not as strongly affected by APOE genotype. We also examined the dendritic morphology of neurons in the medial entorhinal cortex of 3-mo-old TR mice, neurons important to spatial learning functions. We found significantly shorter dendrites and lower spine densities in basal shaft dendrites of E4 mice compared to E3 mice, consistent with spatial learning and memory deficits in E4 animals. These findings suggest that human APOE-14 may affect cognitive function and neuronal morphology early in life.

Intraneuronal accumulation of β-amyloid (Aβ)42 is one of the earliest pathological events in humans and in animal models of Alzheimer's disease (AD). Apolipoprotein E 4 (APOE4) is the major identified genetic risk factor for late-onset AD, with Aβ deposition beginning earlier in apoE4-positive subjects. To directly determine the effects of APOE genotype on intraneuronal accumulation of Aβ1-42 at the onset of AD pathogenesis, we introduced lentiviral Aβ1-42 into the cortex of APOE targeted replacement (TR) mice at the age of 8-9 months. We demonstrated a significant isoform-dependent effect of human APOE, with dramatically enhanced intracellular Aβ1-42 deposits in the cerebral cortex of APOE4-TR mice 2 weeks after injection. Double-immunofluorescent staining showed that intracellular accumulation of lentiviral Aβ1-42 was mainly present in neurons, localized to late endosomes/lysosomes. This intraneuronal accumulation of Aβ1-42 correlated with increased tau phosphorylation and cell death in the ipsilateral cortex around the injection site. Aβ1-42 was also observed in microglia, but not in astrocytes. Quantitative analysis revealed more neurons with Aβ1-42 while less microglia with Aβ1-42 nearest to the injection site of Aβ1-42 lentivirus in APOE4-TR mice. Finally, apoE was present in neurons of the ipsilateral cortex of APOE-TR mice at 2 weeks after lentivirus injection, in addition to astrocytes and microglia in both the ipsilateral and contralateral cerebral cortex. Taken together, these results demonstrate that apoE4 tips the balance of the glial and neuronal Aβ toward the intraneuronal accumulation of Aβ.

High levels of the amyloid-beta (Aβ) peptide have been shown to disrupt neuronal function and induce hyperexcitability, but it is unclear what effects Aβ-associated hyperexcitability may have on tauopathy pathogenesis or propagation in vivo. Using a novel transgenic mouse line to model the impact of human APP (hAPP)/Aβ accumulation on tauopathy in the entorhinal cortex-hippocampal (EC-HIPP) network, we demonstrate that hAPP overexpression aggravates EC-Tau aggregation and accelerates pathological tau spread into the hippocampus. In vivo recordings revealed a strong role for hAPP/Aβ, but not tau, in the emergence of EC neuronal hyperactivity and impaired theta rhythmicity. Chronic chemogenetic attenuation of EC neuronal hyperactivity led to reduced hAPP/Aβ accumulation and reduced pathological tau spread into downstream hippocampus. These data strongly support the hypothesis that in Alzheimer's disease (AD), Aβ-associated hyperactivity accelerates the progression of pathological tau along vulnerable neuronal circuits, and demonstrates the utility of chronic, neuromodulatory approaches in ameliorating AD pathology in vivo.

Recently, food processors and technologists have shown a great interest in the isolation, identification, and purification of natural pigments, including carotenoids, due to their nutritional value, their use as colorants, and their potential as health aids. This unit describes a practical way of extracting, isolating, and purifying carotenoids from plant materials. The method is based mainly on the natural form in which the carotenoids are found (esterified or free) and to some extent on their polarity and/or solubility in the solvents used. Common and readily available solvents and laboratory equipment are suggested. The extraction and isolation of three groups of carotenoids of different polarity are described in Basic Protocol 1. A method for prepurifying carotenoids using crystallization is described in Basic Protocol 2. Carotenoids may be purified further by chromatographic techniques (UNIT F2.3) and characterized (UNITS F2.2 & F2.4). Support Protocols 1 and 2 describe the preparation of the sample before extraction. This process consists mainly of removing water from the sample followed by sample grinding or homogenizing. NOTE: Carotenoid pigments should be protected from light, oxygen, and heat (see Critical Parameters and Troubleshooting). BASIC PROTOCOL 1 SOLVENT EXTRACTION AND ISOLATION OF CAROTENOIDS This protocol begins with the extraction of a dehydrated sample. It continues with a saponification scheme to initiate the isolation of the carotenoid mixture. During saponification, the esters are hydrolyzed and the free pigments released. Then, to continue the isolation, column chromatography is suggested as a simple and fast means of separating the three main groups of carotenoids based on their different polarities. Materials Dehydrated plant material (see Support Protocols 1 and 2) Extractant: 1:1 (v/v) hexane/acetone or hexane alone Saponifying solution: 40% (w/v) KOH in methanol (cool to room temperature before bringing up to volume) Salting-out solution: 10% (w/v) Na 2 SO 4 Na 2 SO 4 , anhydrous (powder form) Adsorbent (see recipe) Carotene eluant: 4% (v/v) acetone in hexane Monohydroxy pigment (MHP) eluant: 1:9 (v/v) acetone/hexane Dihydroxy pigment (DHP) eluant: 1:8 (v/v) acetone/hexane 500-ml extraction vessel Explosion-proof shaft mixer (e.g. model SIU04X; Lightnin) Whatman no. 42 filter paper Rotary evaporator (e.g., Büchi Rotavapor, Brinkmann Instruments) attached to vacuum pump, ≤55°C 56°C water bath 125-ml separatory funnel 600 × 40-mm chromatography column Glass wool Vacuum filtration device 1-liter filtration flasks Contributed by Gustavo A. Rodriguez

13High levels of the amyloid-beta (Aβ) peptide have been shown to disrupt neuronal function and 14 induce hyperexcitability, but it is unclear what effects Aβ-associated hyperexcitability may have 15 on tauopathy pathogenesis or propagation in vivo. Using a novel transgenic mouse line to 16 model the impact of hAPP/Aβ accumulation on tauopathy in the entorhinal cortex-hippocampal 17(EC-HIPP) network, we demonstrate that hAPP aggravates EC tau aggregation and accelerates 18 pathological tau spread into the hippocampus. In vivo recordings revealed a strong role for 19 hAPP/Aβ, but not tau, in the emergence of EC neuronal hyperactivity and impaired theta 20 rhythmicity. Chronic chemogenetic attenuation of Aβ-associated hyperactivity led to reduced 21 hAPP/Aβ accumulation and reduced pathological tau spread into downstream hippocampus. 22These data strongly support the hypothesis that in Alzheimer's disease (AD), Aβ-associated 23 hyperactivity accelerates the progression of pathological tau along vulnerable neuronal circuits, 24and demonstrates the utility of chronic, neuromodulatory approaches in ameliorating AD 25 pathology in vivo. 26 Results 84 Aβ-associated acceleration of tau pathology in EC-Tau/hAPP mice in vivo 85Overexpression of mutant hAPP (Swedish/Indiana) in the hAPP/J20 mouse line leads to progressive 86Aβ plaque deposition throughout the hippocampus and neocortex, contributing to aberrant 87 network activity and cognitive deficits (21, 22). To test whether Aβ pathology alters the 88 progression of tau pathology along the EC-HIPP circuit, we generated the hAPP/J20 x EC-Tau 89 mouse line, hereafter referred to as EC-Tau/hAPP (described in Methods). At 16-months, EC-90Tau/hAPP mice exhibit robust amyloid deposition throughout the EC and HIPP using 6E10 91( Figure 1B), with no change in Aβ deposition compared to age-matched hAPP mice sampled. 92Diffuse amyloid accumulation made up the majority of the pathology, though small, compact Aβ 93 plaques and large, dense-core Aβ plaques were also present (arrows, Figure 1B). Mice 94 expressing hTau alone (EC-Tau) did not exhibit 6E10 immunoreactivity ( Figure 1A). 95Immunostaining for MC1 revealed a dramatic increase in abnormal, misfolded tau within the 96 somatodendritic compartments of EC and HIPP neurons of EC-Tau/hAPP mice compared to 97 EC-Tau littermates ( Figure 1C-D). This increase was over three-fold higher in EC cells (EC-98 Tau/hAPP: 131.00 ± 19.38 vs EC-Tau: 40.52 ± 7.24) and over ten-fold higher in DG granule 99 cells (EC-Tau/hAPP: 359.20 ± 109.40 vs EC-Tau: 30.67 ± 14.88) ( Figure 1E), suggesting that 100 hAPP/Aβ expression in the EC-HIPP network accelerates tau pathology along the classical 101 perforant pathway. MC1 immunostaining in 10-month EC-Tau/hAPP mice revealed mostly 102 diffuse tau in neuropil throughout the EC and in axons terminating in the middle-and outer-103 molecular layers of the DG ( Figure 1F). No somatodendritic MC1+ staining was detected in 104HIPP subregions at this age. By 16-months, it was clear that tau aggregation had not ...